Role of alphaBI5 and alphaBT162 residues in subunit interaction during oligomerization of alphaB-crystallin.
OBJECTIVE
To determine whether the residues in the NH(2)- and COOH-terminal extensions interact with one another during oligomerization of alphaB-crystallin.


METHODS
Site-directed mutagenesis was used to mutate alphaBI5 and alphaBT162 residues to Cys. The recombinant I5C and T162C proteins were expressed in Escherichia coli cells and purified using chromatographic techniques. These proteins were analyzed by SDS-PAGE and mass spectrometry and characterized by multi-angle light scattering and circular dichroism (CD) spectroscopy methods. Fluorescence resonance energy transfer (FRET) assay was used to determine the interaction between the subunits.


RESULTS
Dimer formation was observed in both alphaBI5C and alphaBT162C in storage at 4 degrees C. During air oxidation at room temperature, alphaBT162C formed dimers to a greater extent than alphaBI5C. The average molar masses, secondary structures, and chaperone-like activities of the reduced forms of I5C and T162C were comparable to that of wild type alphaB-crystallin. The oligomeric assembly of reduced forms of I5C and T162C appeared homogenous under JEOL 1200EX Electron microscope whereas the oxidized proteins appeared as irregular aggregates. FRET assay demonstrated interactions between alphaBI5C-alphaBI5C and alphaBT162C-alphaBT162C. However, there was no evidence of an interaction between alphaBI5C and alphaBT162C residues during oligomerization.


CONCLUSIONS
This study suggests that residues from the NH(2)- and COOH-terminal regions in alphaB-crystallin interact with residues from the corresponding regions of another subunit, but there exists no interaction between the residues at the COOH-terminal extension region and the residues at the NH(2)-terminal region.
Murugesan
Raju
R
Department of Ophthalmology, University of Missouri, Columbia, MO 65212, USA.
Santhoshkumar
Puttur
P
Sharma
K Krishna
KK
D000596:Amino Acids / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008970:Molecular Weight; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014364:Tryptophan; D038203:alpha-Crystallin B Chain / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure
D000596:Amino Acids; D000817:Anilino Naphthalenesulfonates; D050505:Mutant Proteins; D021122:Protein Subunits; D038203:alpha-Crystallin B Chain; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D014364:Tryptophan